scholarly journals HIV-1 capsid binds host cofactors to mediate nuclear import

2014 ◽  
Vol 70 (a1) ◽  
pp. C120-C120
Author(s):  
Amanda Price ◽  
David Jacques ◽  
Sebastian Essig ◽  
Tom Elliott ◽  
Upul Halambage ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Binding Sites ◽  
Essential Role ◽  
Antiretroviral Drugs ◽  
Nuclear Pore ◽  
The Core ◽  
Early Stages ◽  
Important Target ◽  
Hexameric Form ◽  
Hiv 1

The capsid (CA) protein of HIV-1, which forms the core of the virus, has been shown to have an increasingly important role in the early stages of the virus lifecycle, in particular during reverse transcription and nuclear import. We recently solved the structure of a fragment of the human cofactor CPSF6 in complex with the N-terminal domain of HIV-1 CA, revealing a previously unknown interface used by the virus to recruit CPSF6, which is required for the virus to successfully complete the early stages of its lifecycle. Using a recently developed hexameric unit of CA, we have solved the structure of the CPSF6 peptide with CA in a context that more closely resembles an intact CA lattice. This has revealed that CPSF6 contacts HIV-1 CA using an additional second site only present in the hexameric form of CA. Furthermore, we have now solved the structure of a fragment of NUP153 (an HIV-1 cofactor that is integral to the nuclear pore) in complex with hexameric CA and discovered that this also forms contacts specific to hexameric CA. Moreover, the binding sites for CPSF6 and NUP153 on CA overlap at one crucial residue, which is remarkably mimicked by two drugs independently discovered to bind at this same site. Together, these data provide evidence for an essential role for CA in HIV-1 infection, and highlights CA as an important target for antiretroviral drugs.

scholarly journals Selective Disruption of Nuclear Import by a Functional Mutant Nuclear Transport Carrier

2000 ◽  
Vol 151 (2) ◽  
pp. 321-332 ◽  
Author(s):  
Cynthia M. Lane ◽  
Ian Cushman ◽  
Mary Shannon Moore
Keyword(s):  
Nuclear Import ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Nuclear Transport ◽  
Dominant Negative ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Binding Studies ◽  
Transport Carrier ◽  
Docking Sites

p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) α and β1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-β1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-β1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-β1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

scholarly journals HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

2021 ◽  
Author(s):  
Anabel Guedán ◽  
Callum D Donaldson ◽  
Ophélie Cosnefroy ◽  
Ian A Taylor ◽  
Kate N. Bishop
Keyword(s):  
Reverse Transcription ◽  
Integration Site ◽  
Productive Infection ◽  
Nuclear Pore ◽  
Virus Infectivity ◽  
Nuclear Speckles ◽  
Nuclear Entry ◽  
The Core ◽  
Fold Reduction ◽  
Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

scholarly journals Importin β contains a COOH-terminal nucleoporin binding region important for nuclear transport

2003 ◽  
Vol 162 (3) ◽  
pp. 391-401 ◽  
Author(s):  
Janna Bednenko ◽  
Gino Cingolani ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Binding Sites ◽  
Nuclear Transport ◽  
Terminal Region ◽  
Nuclear Pore ◽  
Binding Region ◽  
Similar Extent ◽  
Importin Α ◽  
Import Receptor

Proteins containing a classical NLS are transported into the nucleus by the import receptor importin β, which binds to cargoes via the adaptor importin α. The import complex is translocated through the nuclear pore complex by interactions of importin β with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin β. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin β to a similar extent (∼50%). An importin β mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin β possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

scholarly journals Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

2016 ◽  
Vol 90 (16) ◽  
pp. 7046-7065 ◽  
Author(s):  
Anjali Verma ◽  
Pavithra Rajagopalan ◽  
Rishikesh Lotke ◽  
Rebu Varghese ◽  
Deepak Selvam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Subtype C ◽  
Viral Promoter ◽  
Factor Binding ◽  
The Core ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTOf the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency.IMPORTANCESubtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined.

scholarly journals Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

1998 ◽  
Vol 141 (1) ◽  
pp. 31-49 ◽  
Author(s):  
Sundeep Shah ◽  
Stuart Tugendreich ◽  
Douglass Forbes
Keyword(s):  
Nuclear Import ◽  
Binding Sites ◽  
Nuclear Pore ◽  
Binding Studies ◽  
Nuclear Pores ◽  
Major Question ◽  
Egg Extracts ◽  
Importin Α ◽  
Xenopus Egg ◽  
Filament Protein

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.

scholarly journals Nuclear Import of HIV-1

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2242
Author(s):  
Qi Shen ◽  
Chunxiang Wu ◽  
Christian Freniere ◽  
Therese N. Tripler ◽  
Yong Xiong
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Integration Site ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
New Paradigm ◽  
Retroviral Infection ◽  
Dividing Cells ◽  
Pore Complexes ◽  
Hiv 1

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial–temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

scholarly journals Correction: Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
Author(s):  
Francesca Di Nunzio ◽  
Anne Danckaert ◽  
Thomas Fricke ◽  
Patricio Perez ◽  
Juliette Fernandez ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Pore ◽  
Hiv 1

scholarly journals Cone-shaped HIV-1 capsids are transported through intact nuclear pores

2020 ◽  
Author(s):  
Vojtech Zila ◽  
Erica Margiotta ◽  
Beata Turonova ◽  
Thorsten G. Müller ◽  
Christian E. Zimmerli ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Light And Electron Microscopy ◽  
Productive Infection ◽  
Nuclear Pore ◽  
Major Health ◽  
Replication Complex ◽  
Empty Capsid ◽  
Immunodeficiency Virus ◽  
Subtomogram Averaging ◽  
Hiv 1

AbstractHuman immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

scholarly journals Author response: HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex

2018 ◽  
Author(s):  
David Alejandro Bejarano ◽  
Ke Peng ◽  
Vibor Laketa ◽  
Kathleen Börner ◽  
K Laurence Jost ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Author Response ◽  
Nuclear Pore ◽  
Hiv 1
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